(W1230-09-60) A Preclinical Method to Quantify Complex Formulation Interactions with Serum Proteins and Target Tissues

Purpose: Breast cancer is the most common cancer in women, accounting in the US for over 30% of new cancer cases and more than 42000 deaths in 2025. The heterogeneity of breast cancer types and interindividual variability make the development of targeted therapies, such as nanoparticles and biologics, highly complex, with 90% of new drugs failing in clinical trials. Among the factors influencing mortality rates, tumor aggressiveness, and progression, the tumor extracellular matrix (ECM) has recently emerged as a key player. The ECM modulates the tumor microenvironment, regulating signaling molecule exchange, supporting tumor cell proliferation, while also providing mechanical structure. Variations in ECM structure may hinder the efficacy of targeted therapies by limiting drug uptake at the tumor site. The characterization of the physicochemical properties of both the ECM and the drug formulation is therefore essential for new targeted therapy development. In this study, we developed a novel method to characterize and quantify the physicochemical properties of the breast cancer ECM and its interactions with nanosized drug delivery vehicles in the presence of systemic protein.

Methods: Liposomes were prepared using the thin-film hydration technique, followed by sonication. Doxil-like liposomes were prepared varying DSPE-PEG content, molecular weight, and terminal groups. Contact angles of water, liposomes, and albumin (BSA)-liposome mixtures were measured on glass slides using a customized optical goniometer. De-identified breast tissue sections were firstly deparaffinized and then decellularized using 3 freeze-thaw cycles in water. ECM were incubated with liposomes or liposomes-albumin mixture for 2 min, rinsed with DI water, and contact angle was measured again using our customized instrument. To remove liposomes and albumin from ECM surfaces, tissues were treated with Ethanol 1% at 55 °C for 2 minutes, soaked in SDS for 1 min, and rinsed several times with DI water.

Results: Doxil-like liposomes decreased water contact angle proportionally as the suspension concentration increased. Pegylation was the principal factor contributing to this behavior, with liposomes 100% and 150% PEG causing a remarkable decrease in contact angle. Reducing PEG’s amount, an increase in contact angle was observed. PEG-free liposomes had no significant surface activity. Higher PEG molecular weight and OCH3, -COOH end-caps caused the highest contact angle reduction and spreading. The addition of albumin in the liposome formulation reversed this effect. Breast cancer ECM showed higher contact angles compared to normal adjacent tissues, especially for advanced tumor stages, revealing an increase in hydrophobicity as the tumor progressed. This effect was reversed when tissues were incubated with liposomes, with pegylated liposomes showing a drastic decrease in water contact angle, particularly pronounced in metastatic and advanced cancer tissues. Albumin interferes with liposomes-tissue interaction, reducing their ability to adhere to the ECM surface.

Conclusion: Our data highlight and quantify the importance of PEG as a surface molecule in the interactions measured by the contact angle between liposomes, tumor ECM, and proteins associated with reduced efficacy. These findings align with clinical trials of Doxil®, where the inclusion of PEG was critical to achieve therapeutic efficacy. Interestingly, liposome surface action measured by contact angle was reversed when albumin was incorporated into the formulation, suggesting a strong surface interaction between the two. In addition, the relative hydrophobicity of tumor ECM compared to nearby healthy ECM was also reversed when tissues were incubated with liposomes. Overall, our method is sensitive to formulation characteristics known to be clinically relevant, enabling the development of preclinical testing methods to predict clinical efficacy utilizing patient biopsies towards applications such as individualized medicine and targeted therapies.

References: (1) Huang, J., Zhang, L., Wan, D. et al. Extracellular matrix and its therapeutic potential for cancer treatment. Sig Transduct Target Ther 6, 153 (2021).
(2) Mishra I, Garrett M, Curry S, Jameson J, Kastellorizios M. Effect of Composition and Size on Surface Properties of Anti-Cancer Nanoparticles. Int J Mol Sci. 2023 Aug 30;24(17):13417
(3) Rincon, J. &. (2023). Novel Method to Obtain Contact Angles of Tumor Biopsies. ACS omega, 26965–26972.
(4) Henke E.; Nandigama R.; Ergun S. Extracellular Matrix in the Tumor Microenvironment and Its Impact on Cancer Therapy. Front. Mol. Biosci. 2020, 6, 160.

Acknowledgements: This work is partially supported by National Institute on Minority Health and Health Disparities (Grant #2U54MD006882-06) and by the Pharmaceutical Research and Manufacturers of America Foundation, 2019 Research Starter Grant in Pharmaceutics.
Presenter supported by Doctoral Scholars in Cancer Research (DSCR), under the HSC-Scholars in Cancer Research Program sponsored by Cancer Prevention and Research Institute of Texas (CPRIT)

Fig. 1a: Contact angles of Albumin solutions at different concentration (0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20 mg/ml) measured on glass. A reduction in contact angle was observed at the concentration of 1 mg/ml with our instrument and was overall consistent for higher concentrations.
Fig. 1b Contact angles of Doxil-like liposomes with increasing DSPE-PEG OCH₃ content (0%, 25%, 50%, 75%, 100%, 150%) in their composition compared with the contact angle of Albumin-liposomes mixtures for the same liposomal compositions. A reserved trend was observed from that of PEGylated liposomes when albumin was added to the suspension.


Fig. 2: Variation in contact angle observed in decellularized breast tissue samples, including Normal Adjacent Tissue (NAT), Cancer Adjacent Tissue (CAT), and breast tumor tissues (ductal carcinoma and lobular carcinoma). Each data point represents the average contact angle of all samples of the same tissue type present on the slides. An increase in hydrophobicity was observed when progressing from healthy to cancerous tissues, with metastatic tissues exhibiting the highest contact angles